The chief objective of this investigation is to clarify the mechanism of action of cardiac cytochrome c oxidase. The interrelationships of cytochromes a and a3 and copper in the enzyme will be studied in heart muscle and in a microbial system using spectrophotometric and electron paramagnetic resonance (EPR) techniques. The reaction of cytochrome oxidase with O2 will be investigated to identify intermediates in this reaction by using a new flow laser-flash technique. The structure of Pseudomonas cytochrome oxidase in crystalline form will be studied using an electron diffraction technique. Because one of the heme groups heme d1, of Pseudomonas cytochrome oxidase can be removed and reconstituted, heme groups (iron-chlorins) closely related to heme d1 in structure will be synthesized and used as substitutes for heme d1. The purpose of these experiments is to obtain information about the requirements for reaction of heme groups with O2.